1. Field of the Invention
The invention pertains to methods and dispensing apparatuses providing precise, very small quantities of fluids.
2. Description of the Prior Art
It is important in a variety of industries, such as medical diagnostics, biotechnology, and scientific instrumentation, to accurately dispense very small drops of fluids. Furthermore, it is desirable to be able to program the volume of the drops so that the amount delivered will be precise and accurate while at the same time minimizing the amount of a sample required for the dispenser. Some examples of small volume dispensing devices are described in U.S. Pat. Nos. 5,366,896; 5,919,706; 5,927,547; 5,958,342; 5,998,218; 6,083,762; 6,090,348; and 6,100,094. Ink jet printer devices represent an example of a technology area where systems and methods for dispensing small volumes of fluid have been developed. However, the ink jet printer devices suffer from the drawback that they often require several microliters of fluid to prime the dispenser passage; even if only sub-nanoliter sized droplets are dispensed. In many technologies, it would be advantageous to be able to aspirate a volume of about a nanoliter or less without needing to pick up larger amounts. This problem is especially acute in forensic sciences and in biotechnology where only limited quantities of sample are available.
One difficulty with dispensing small volumes of fluid is the necessity of a tip with a small radius. The small radius results in large internal pressures that prevent the fluid from flowing easily from the tip. To overcome this limitation, other systems expel fluids by forcibly ejecting the droplets at a high velocity. However, these systems suffer from accuracy problems. It would be desirable for a system to be able to aspirate and deliver small volumes without being susceptible to clogging, while still maintaining a high level of accuracy.
It is an object of this invention to overcome the limitations of the prior art, and to provide a highly accurate dispensing device and method which allows dispensing controllable droplets of sub nanoliter size without requiring relatively large priming volumes.
This invention contemplates the use of a pipette or probe that includes a working fluid, an air gap, and a sample fluid in the dispensing tip. The pipette or probe is used to first retrieve a quantity of the sample fluid at the tip. In the retrieval, the working fluid and air gap rise within the pipette or probe, and the sample fluid fills the end of the tip. Then, the invention contemplates dispensing a sessile drop onto a substrate. Preferably, a small portion of the sample remains in the end of the tip, and the tip contacts the outer periphery of the sessile drop. A camera or other imaging device is used to measure the diameter and height of the fluid. This information is then used to calculate the volume inside the tip while it is still in contact with the sessile drop. Then, precise amounts of fluid in selectively variable quantities are drawn back up into the pipette tip from the sessile drop. The tip is then moved to a desired dispensing location, and the desired sample fluid is expelled from that remaining in the tip.
Physically, movement of fluid into a very narrow channel pipette or probe tip is difficult to achieve. The technique utilized in this invention promotes the ability to siphon up sample fluid by different mechanisms. First, creating a sessile drop physically provides a fluid with a surface of curvature that will promote siphoning. Laplace""s rule states that the pressure across an interface is proportional to interfacial (surface) tension and inversely proportional to radius of curvature. The small radii inside pipette tips, therefore, leads to large pressures. Second, the liquid surface does not move smoothly over the pipette inside surface because the surface is not energetically constant (i.e., even) and because of what is known as contact angle hysteresis (advancing angles are not equivalent to receding angles). For these reasons, fluid motion is not steady; rather it is stop and start, and may often be referred to as stick/slip. Combined with the high and variable pressures from LaPlace""s rule, it is extremely difficult to directly draw or dispense a specific amount from a continuum of liquid.
The genesis of the invention is that the dispense volume is separated from the larger supply volume in a preparatory step before the actual dispense phase. The dispense volume is contained in the end of a capillary tube, separated by an airgap from any system liquid in the pumping system. The exact volume of dispense liquid is set by adjusting the dispense volume while the tip is immersed in a sessile drop of the same liquid. The sessile drop has much larger radii of curvature than the liquid in the tip, and these larger radii lower the interfacial pressure following Laplace""s rule.
When one ponders any dispense operation, there are the following two phases: setting the volume to dispense, and detaching the dispensed volume from the remainder of the liquid. This is so basic that it is ordinarily not enunciated. Ordinarily both functions are performed by the same means, and often at the same or very similar times. This is true whether one considers classical syringe pumps or modern ink-jet printer mechanisms. The current invention takes a different and unique approach in that it separates the two phases. As a simplistic analogy, the first phase can be thought of as a xe2x80x9crulerxe2x80x9d to set or measure the volume and the second phase can be thought of as xe2x80x9cscissorsxe2x80x9d to separate the volume from its parent or source. This invention separates the ruler from the scissors. Furthermore, there are two kinds of scissors used.
First Phase: The ruler function is performed by video image analysis while the tip is immersed in the sessile drop. The airgap that is set above the liquid in the tip both permits the accurate volume determination through the transparent capillary and is preparatory to the detachment phase. The detachment phase is now subdivided into two portions. This is important. The first occurs when the tip is pulled up from the sessile drop. The tip breaks clean of the sessile drop because the tip is very small. The same Laplace pressures that bedevil us elsewhere ensure that the tip comes out clean, without a hanging pendant drop. The dispense volume is exactly what is inside the tip and what was measured by video analysis. The impetus for the first portion of the detachment is the motor driving the tip up and down. Most importantly, it is not the pump proper. The pump is not able to perform this detachment. So instead, the motorized Z stage separates the xe2x80x9cchildxe2x80x9d volume (the small volume of sample to be dispensed) from the xe2x80x9cparentxe2x80x9d volume (the volume of the sessile drop).
Second Phase: The pump plays a role in the second portion, which occurs later when the tip is disposed over the target. The pump pushes the dispense liquid out of the tip, either rapidly or slowly, as the user desires. There are applications for all kinds of dispense momentums, or momenta.
In summary, the Z motor provides the scissors between the liquid in the tip and the liquid in the sessile drop. The pump provides the scissors between the airgap and the dispensed volume over the target. The larger radius of the sessile drop used in this invention lowers the pressures, and the high frequency vibrations contemplated by this invention breaks loose the stick/slip motion. In addition, this invention contemplates providing vibrations to the pipette or probe tip. This can be achieved by acoustic or mechanical means (e.g., a piezo ceramic element may be driven to sequentially compress and de-compress the working fluid).
The method and apparatus of this invention are adaptable to robotic placement of very small fluid samples at precise locations. This may have application in certain antibody and DNA detection chips, as well as in a variety of other applications. For example, by having precise quantities of fluid containing an antigen or antibody or single stranded DNA or any other molecular entity placed on a chip or other substrate, it would be possible to optically assess weight differentials which are the result of selective bonding or hybridizing reactions.